Lignans are major constituents of plant extracts and have important
pharmacological effects on mammalian cells. Here we showed that
pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) from Valeriana
officinalis induced calcium mobilization and cell migration through the
activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation
of mouse embryo fibroblast (MEF) cells with 10 [micro]M PDG resulted in
strong stimulation of MEF cell migration and the [EC.sub.50] was about
2[micro]M. Pretreatment with pertussis toxin (PTX), an inhibitor of
[G.sub.i] protein, completely blocked PDG-induced cell migration
demonstrating that PDG evokes MEF cell migration through the activation
of the [G.sub.i]-coupled receptor. Furthermore, pretreatment of MEF
cells with Kil6425 (10 [micro]M), which is a selective antagonist for
[LPA.sub.1] and [LPA.sub.3] receptors, completely blocked PDG-induced
cell migration. Likewise, PDG strongly induced calcium mobilization,
which was also blocked by Ki16425 in a dose-dependent manner. Prior
occupation of the LPA receptor with LPA itself completely blocked
PDG-induced calcium mobilization. Finally, PDG-induced MEF cell
migration was attenuated by pretreatment with a phosphatidylinositol
3-kinase (P13K) inhibitor such as LY294002. Cells lacking downstream
mediator of PI3K such as Aktl and Akt2 (DKO cells) showed loss of
PDG-induced migration. Re-expression of Aktl (but not Akt2) completely
restored PDG-induced DKO cell migration. Given these results, we
conclude that PDG is a strong inducer of cell migration. We suggest that
the pharmacological action of PDG may occur through the activation of an
LPA receptor whereby activation of PI3K/Akt signaling pathway mediates
PDG-induced MEF cell migration.

The crude extract of Valeriana officinalis root is used for
traditional medicine as a mild sedative and tranquilizer in many
countries (Houghton, 1999). The genus Valeriana consists of 200 species
and belongs to the family of Valerianaceae which is widely distributed
throughout the world. Valeriana officinalis has been used as an
anticonvulsant, for its hypnotic effects, and as an anxiolytic in some
countries (Carlini, 2003). In addition, it has been demonstrated that
valerian has mild central nervous system (CNS)-depressant effects in
mice (Leuschner et al., 1993) as well as in some clinical studies
(Fugh-Berman and Cott, 1999). Valerian also has neuroprotective effect
by inhibiting excess calcium influx (Malva et al., 2004). However, the
molecular mechanism of the CNS-related action of valerian is still
unclear.

Valeriana officinalis consist of over 150-200 chemical constituents
including flavonoids and lignans. A flavo-noid such as 6-methylapigenin
in Valeriana officinalis has a benzodiazepine binding site (Wasowski et
al., 2002) and has sedative as well as sleep-enhancing activity (Marder
et al., 2003). Lignans from Valeriana officinalis have antioxidative and
vasorelaxant effects (Piccinelli et al., 2004). Structurally,
pinoresinol is one of the simplest lignans; it is a dimer of coniferyl
alcohol and is frequently present in woody or fibrous plants. Recently,
it has been reported that 8-hydroxypinoresinol has affinity for
5-[HT.sub.1A] receptors at low micromolar concentrations (Bodesheim and
Hoelzl, 1997). In addition,
4'-O-[beta]-D-glucopyranosyl-9-0-(6"-deoxysaccharosyl)olivil
has been demonstrated to have affinity for [A.sub.1] adenosine receptors
(Schumacher et al., 2002). Although several reports have demonstrated
the ability of pinoresinol modulates several CNS receptors, stimulatory
and inhibitory effects of other cell physiology are largely unknown.

Chemotaxis or directional cell migration plays a critical role in
tissue development, immune response, and tissue repair. In pathological
conditions, aberrant signaling leads to enhanced chemotactic migration
such as tumor metastasis (Lauffenburger and Horwitz, 1996). Cell
migration is initiated by the activation of cell surface receptors such
as growth factor receptors and G protein-coupled receptors (GPCR),
leading to the activation of PI3K/Akt signaling pathways, calcium
mobilization, and activation of small G proteins.

Powdered Valeriana officinalis (VO) roots were obtained from
Frontier Company (2000 Frontier, IA 52318, Norway). Samples were
deposited at the University of Mississippi, National Center for Natural
Products Research. The finely ground roots of valerian (1 kg) were
successively refluxed with hexane, chloroform and methanol overnight.
After cooling, the suspension was filtered, and the methanolic solution
was evaporated under reduced pressure. The brown, oily residue (10% of
original weight) was imbedded in silica.

Extraction and spectral analysis of
pinoresinol-4,4'-di-O-[beta]-D-glucoside

The MeOH extract (52 g) was evaporated in vacuo and chromatographed
on a silica gel (40 [micro]m, J.T. Baker, NJ, USA) column (70 x 8.0 cm)
with a step gradient 5%, 10%, 15% MeOH in chloroform (each 21) to get 15
fractions. Fractions were collected and checked by TLC on silica gel 60
[F.sub.254] and reverse phase (RP) glass plates. Fraction 8 (1444 mg)
was separated on a Sephadex 20 column (60 x 3.0 cm) with MeOH to give a
pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) (234 mg). The
chemical structure of PDG was verified by LC-MS (Bruker BioApex FT mass
spectrometer) and NMR analysis (Bruker DRX 400 spectrometer). Optical
rotations were recorded on a JASCO DIP-370 digital polarimeter. IR
spectra were recorded on an AATI Mattson Genesis Series FTIR. NMR
spectra ([.sup.1]H, [.sup.13]C) were recorded in [CDC1.sub.3] on a
Bruker DRX 400 spectrometer operating at 400MHz for [.sup.1]H and 100
MHz for [.sup.13]C, running gradients, and using residual solvent peaks
as internal references. High-resolution mass spectra were recorded on a
Bruker BioApex FT mass spectrometer.

MEF cells were isolated the same way as described previously (Yun
et al., 2008). Embryos were dissected from pregnant
Aktl/PKB[[alpha].sup.[+/-]]; Akt2/PKB[[beta].sup.[+/-]] females that had
been bred to Aktl/PKB[[alpha].sup.[+/-]]; Akt2/PK[[beta].sup.[+/-]]
males. The yolk sacs, heads, and internal organs were isolated and used
for genotyping by RT-PCR. Carcasses were treated with trypsin-EDTA for
30 min at 37 [degrees]C, and clumps of cells were disrupted by chopping
with scissors. After centrifugation, the cells were re-suspended in
culture medium (DMEM supplemented with 10% FBS and antibiotics) and
maintained at 37 [degrees]C in 5% [CO.sub.2]. Primary cells were
immortalized by continuous culturing for 30 passages.

Retroviral infection

Generation of viral supernatant was done as previously reported
(Yun et al., 2008). Briefly, ecotropic BOSC23 cells were transiently
transfected with pVSV/G and pGag/pol, pantropic retroviral packaging
constructs, and retroviral vector containing Aktl/PKB[alpha],
Akt2/PKB[beta]. Cell-free viral supernatants were mixed with one volume
of complete medium in the presence of 8 [micro]g/ml polybrene and used
to infect immortalized MEF cells. Cells expressing Akt/PKB are sorted by
flow cytometer (BD Biosciences) and used for the experiments.

MEF cells were grown and serum starved for 6h before plating on the
ChemoTx membrane. Cells were detached with trypsin-EDTA and washed with
serum-free DMEM. For the migration assay, the bottom side of the ChemoTx
membrane was coated with type I collagen for 30 min, and 2 x [10.sup.4]
serum-starved cells (in 50 [micro]l) were placed on the top side of the
ChemoTx membrane. Migration was induced by submerging the ChemoTx
membrane in serum-free medium either in the presence or in the absence
of chemotactic attractants for 3 h. The ChemoTx membrane was fixed with
4% paraformaldehyde, and non-migratory cells on the top side of the
membrane were removed by gently wiping with a cotton swab. The membrane
was stained with DAPI, and migrating cells were counted under the
fluorescence microscope at 10 x magnification (Axiovert 200).

Results are expressed as the mean[+ or -]S.D. of two independent
experiments (n = 3 for each experiment). When comparing the two groups,
an unpaired Student's t-test was used to address differences.
P-values less than 0.05 were considered significant.

Since LPA-induced MEF cell migration was mediated by PI3K/Akt
signaling pathways (Kim et al., 2008b) and PDG-induced calcium
mobilization was mediated by LPA receptor signaling (Fig. 3), we next
investigated the role of PI3K/Akt signaling pathways in PDG-induced MEF
cell migration. As shown in Fig. 4A, PDG-induced MEF cell migration was
completely blocked by pretreatment of LY294002 (10 [micro]M), which is a
PI3K inhibitor; however, inhibition of ERK and p38 MAPK pathways did not
affect PDG-induced MEF cell migration (p > 0.05). To delineate the
signaling pathway downstream of PI3K, we examined PDG-induced migration
in cells lacking Akt1 and/or Akt2. As shown in Fig. 4B cells lacking
Akt1 (1KO and DKO) lost their response to PDG-induced migration, whereas
the loss of Akt2 (2KO) did not significantly affect it. Finally, either
Akt1 or Akt2 was re-introduced into Akt1 and Akt2 double knock-out (DKO)
cells (Fig. 4). Ectopic expression of Akt1 or Akt2 was about 100-fold
higher than endogenous Akt1 and Akt2 in wild-type cells. DKO cells
completely restored PDG-induced migration by re-expression of Akt1 but
not by Akt2 (Fig. 4D).

[FIGURE 4 OMITTED]

Discussion

Plant extracts have a variety of physiological effects and have
been used in traditional medicine since ancient times. This is
especially true for the crude extract of Valeriana officinalis which has
CNS-depressant and neuroprotective effects. Lignans are major
constituents of crude plant extracts and pinoresinol is structurally one
of the simplest lignans. Especially, Eucommia ulmoides extract has been
used for antihypertensive, anti-inflammatory, and sedative activity in
which lignans are major responsible components mediating those effects.
Recent experiments have demonstrated that lignans have binding affinity
for serotonin and adenosine receptors (Bodesheim and Hoelzl, 1997;
Schumacher et al., 2002). However, the exact biological function and
molecular mechanism of pinoresinol in mediating cell migration and
calcium mobilization, which are important process for neuronal activity,
cardiovascular function, and inflammation, are unknown. In the present
study, we provide novel insight that
pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) is a strong
modulator for cell migration and calcium mobilization.

Virtually, we have screened 45 compounds from Valeriana officinalis
and Schisandra chinensis for cell migration and vasorelaxation. Gomisin
A from Schisandra chinensis was identified as vasorelaxant (Park et al.,
2007). Although it has been reported that lignans from Valeriana
prionophylla have vasorelaxant activity at high molar concentration
(10-100 [micro]M), low molar concentration of PDG (5 [micro]M) did not
have vasorelaxant activity (data not shown). However, PDG strongly
stimulated cell migration and calcium mobilization at 5 [micro]M,
indicating that migration and calcium mobilization rather than
vasorelaxation are more susceptible to PDG stimulation. Therefore, we
provide novel physiological function of PDG as a strong modulator of
cell migration and calcium mobilization, which are important for the
inflammation and neural function.

Several lines of evidences support that PDG-depen-dent MEF cell
migration is mediated by LPA receptor subtype. First, the possible
involvement of receptor tyrosine kinase could be excluded since
PDG-dependent cell migration was not affected by pretreatment of
receptor tyrosine kinase inhibitor (Fig. 2A). However, PDGF-induced cell
migration was completely blocked by receptor tyrosine kinase inhibitor.
Second, PDG-induced cell migration was blocked by inactivation of a
[G.sub.i]-coupled receptor using pertussis toxin (Fig. 2A),
demonstrating that a PDG receptor is [G.sub.i]-coupled. Since LPA
receptor is also coupled to [G.sub.i] protein (Hilal-Dandan et al.,
2004), LPA-induced cell migration was completely blocked by pretreatment
of pertussis toxin (Fig. 2A). Third, it has been shown that Ki16425 is a
selective inhibitor of [LPA.sub.1] and [LPA.sub.3] (Ohta et al., 2003),
and Ki16425 blocked cell migration induced by both PDG and LPA (Fig.
2B). Therefore, it is possible that PDG-dependent cell migration may be
mediated by the activation of an LPA receptor subtype at least in part.

Calcium mobilization is a major downstream response of LPA receptor
activation (Lee et al., 2007). In correlation with this, stimulation of
MEF cells with LPA strongly evoked calcium release (Fig. 3A). PDG also
induced calcium mobilization, although the maximum response was about
50% of LPA-induced calcium mobilization. This may be due to the
subtype-selective activation of LPA receptor. For example, inactivation
of [LPA.sub.1] and [LPA.sub.3] by high concentration of Ki16425 (10
[micro]M) resulted in complete blocking of PDG-induced calcium
mobilization, whereas LPA-induced calcium mobilization was partly
blocked at the same concentration of Ki16425 (Fig. 3C). These results
demonstrate that PDG may selectively induce [LPA.sub.1] and [LPA.sub.3]
to mobilize intracellular calcium. The involvement of an LPA receptor
during PDG-induced calcium mobilization was also confirmed by a
pre-occupation experiment. For instance, pre-occupation of an LPA
receptor by LPA itself completely blocked PDG-induced calcium
mobilization and vice versa (Fig. 3B). It is also noteworthy that
pretreatment of PDG resulted in partial blocking of LPA-induced calcium
mobilization. This may be due to the restricted occupation of
[LPA.sub.1] and [LPA.sub.3] receptors. Therefore, PDG may selectively
exert its effect on [LPA.sub.1] and [LPA.sub.3] receptor subtype and
induces calcium mobilization as well as cell migration.

Previously, we have shown that LPA induces cell migration through
the selective activation of PI3K/Aktl signaling pathway (Kim et al.,
2008b). For example, cells lacking Aktl (but not Akt2) did not stimulate
LPA-dependent migration. LPA-induced DKO cell migration was restored by
re-expression of Aktl but not Akt2. Likewise, PDG-induced cell migration
was mediated by PI3K and abolished in cells lacking Aktl (Fig. 4).
Furthermore, DKO cells restored PDG-induced migration after
re-expression of Aktl but not Akt2. These results indicate that
PDG-induced cell migration is similar to LPA-induced cell migration.
Taken together, we have shown that PDG induces cell migration and
calcium mobilization similar to LPA receptor-dependent pathways.

Although it is unequivocal that PDG-induced cell migration and
calcium mobilization is partly mediated by the activation of LPA
receptor subtype, it is still ambiguous how PDG affects neurological
function. Nonetheless, it is possible that PDG-induced activation of LPA
receptor may affect neurological function thereby inducing sedative and
anxiolytic function. Recent evidences also support the idea that LPA
modulates neurological function. For example, neuropathic pain is
mediated by the generation of LPA (Inoue et al., 2008). More direct
evidence that LPA is involved in the neurological function was
discovered by the evaluation of LPA receptor knock-out mice. Mice
lacking [LPA.sub.1] receptor showed schizophrenia phenotype in which
serotonin efflux was dramatically decreased (Roberts et al., 2005).
Serotonergic neuronal activity is modulated by NMD A receptor (Gartside
et al., 2007), and administration of NMDA receptor antagonist frequently
evokes schizophrenia phenotype (Enomoto et al., 2007). Therefore, both
LPA receptor and NMDA receptor modulate serotonergic neuronal activity
and serotonin efflux. Since serotonin agonists are frequently used for
anxiolytic activity (New, 1990), it is possible that PDG-dependent
modulation of LPA receptor activity may contribute to the mechanism of
anxiolytic activity of PDG. Evaluation of PDG-dependent behavioral
function of mice lacking LPA receptor will provide more insight into
this proposed mechanism.

In conclusion, we have defined the novel molecular mechanism of PDG
action in cell migration and calcium mobilization. We have provided
evidence that PDG could be a novel ligand for [LPA.sub.1] and
[LPA.sub.3] receptors. Studies on the underlying mechanism of PDG on
LPA-related psychiatric function may shed more light on the evaluation
of PDG as an anxiolytic.

Acknowledgements

This study was supported by Technology Development Program for
Agriculture and Forestry (106048031SB010), Ministry of Agriculture and
Forestry, Republic of Korea, and MRC program of MOST/KOSEF
(R13-2005-009) (to S.S.B.).

(a) Department of Pharmacology and MRC for Ischemic Tissue
Regeneration and Medical Research Institute, School of Medicine, Pusan
National University, Busan 602-739, Republic of Korea

(b) College of Natural Resources & Life Sciences, Pusan
National University, Gyungnam 627-706, Republic of Korea

(c) Department of Physiology and MRC for Ischemic Tissue
Regeneration and Medical Research Institute, School of Medicine, Pusan
National University, Busan 602-739, Republic of Korea

(d) National Center for Natural Products Research, Research
Institute of Pharmaceutical Sciences, Department of Pharmacognosy,
School of Pharmacy, The University of Mississippi, University, MS 38677,
USA